Advanced mass spectrometry in proteomics: innovations and applications in immunopeptidomics

Wahle, Maria (2025): Advanced mass spectrometry in proteomics: innovations and applications in immunopeptidomics. Dissertation, LMU München: Faculty of Chemistry and Pharmacy

Abstract

Understanding a biological system begins with describing it. No other technology captures the proteome of a cell as comprehensively as mass spectrometry (MS)-based proteomics. Accordingly, much effort has been dedicated to maximizing the maximum information from each single sample. Progress in MS-based proteomics spans improvements in instrumentation, data acquisition strategies, computational analysis and sample preparation. In this context, I contributed to the development of next generation scan modes on a trapped ion mobility time of flight mass spectrometer (timsTOF) especially suitable for high-throughput or low-input proteomics on this novel MS platform. In addition, I designed a framework for seamlessly translating discovery proteomics into targeted assays using a linear ion trap instrumentation, incorporating a workflow for absolute quantification using 15N labeled protein spike-ins. I facilitated the development of a deep leaning based framework uniquely adapted to data independent acquisition (DA) workflows including a standalone model for predicting the presence and MS-behavior of immunopeptides. These components – together with a robust sample preparation pipeline – were integrated into IMBAS-MS, a workflow that I developed for the efficient and high throughput acquisition of immunopeptidomics samples from body fluids. IMBAS-MS identified over 40,000 unique immunopeptides from minimal sample material. Beyond method development, I applied these technological advances to a broad range of biologically relevant projects - from single cell and low input to bulk and clinical proteomics. Two key examples are the development of a cheap multiplexing strategy and its application to low input proteomics, enabling the acquisition of a deep proteome from a fraction of a single hepatocyte and the development of a targeted assay used for biomarker validation in a large clinical cohort investigating neurodegenerative diseases. In summary, this thesis bridges advanced proteomics technologies with immunopeptidomics. It presents contributions across the entire MS-based proteomics pipeline - from sample preparation, acquisition and deep learning assisted data analysis. While rooted in proteomics of tryptic peptides it translates key developments to the field of Immunopeptidomics, providing a robust and scalable framework for widespread adoption for biological and clinical applications.

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: immunopeptidomics, targeted proteomics, clinical proteomics, mass spectrometry, method development, sensitivity
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 540 Chemistry and allied sciences
• Faculties: Faculty of Chemistry and Pharmacy
• Language: English
• Date of oral examination: 27. August 2025
• 1. Referee: Mann, Matthias
• Signature of the printed copy: 0001/UMC 32041